MSP-PCR contamination in NC - (Sep/23/2009 )

I need your suggestion in order to solve one problem which I am facing in my PCR. I am amplifying P15 gene of 101 bp Methylated DNA product using MSP-PCR (Methyl specific PCR). My experiment was going on fine until last week, but past few days I am getting faint positive amplification in my NC (Reagent control: Water instead of DNA). I thought it must be reagent contamination, hence I changed all the reagents including primers and water. But unfortunately I am still getting amplification in my NC. Also I would like to draw your kind attention that when the same reagents were used for amplification of different target gene, there the NC is absolutely clean, thereby raising doubt onIy on P15 primers. I have also given UV treatment to my p15 primers and then again repeated the experiment, but unfortunately I got the same result in NC. Kindly help me solving my problem, which will of great help to me.

Thank you very much in advance.
Cheers
Firoz Ahmad,
Phd student India

-firozsrl-

firozsrl on Sep 23 2009, 05:45 PM said:

Hello!

I need your suggestion in order to solve one problem which I am facing in my PCR. I am amplifying P15 gene of 101 bp Methylated DNA product using MSP-PCR (Methyl specific PCR). My experiment was going on fine until last week, but past few days I am getting faint positive amplification in my NC (Reagent control: Water instead of DNA). I thought it must be reagent contamination, hence I changed all the reagents including primers and water. But unfortunately I am still getting amplification in my NC. Also I would like to draw your kind attention that when the same reagents were used for amplification of different target gene, there the NC is absolutely clean, thereby raising doubt onIy on P15 primers. I have also given UV treatment to my p15 primers and then again repeated the experiment, but unfortunately I got the same result in NC. Kindly help me solving my problem, which will of great help to me.

This sounds like a typical contamination with PCR products. This is really bad, because it means to clean up the whole Pre-PCR lab with sodium hypochlorite solution including pipets, hoods, etc... I had this once and it's definately no fun. If possible try another lab and another set of pipetors for PCR preparation. Do you use pipet tips with aerosol barrier?